Measurement of iron concentration in serum and plasma has been utilized for the diagnosis of anemia, hypo ferric anemia, hepatic cirrhosis and the like, and is one of important measuring items in the field of clinical diagnosis.
As the method of measuring iron concentration, for example, calorimetric analysis using various metallochromic indicators such as, for example, dipyridyl, o-phenanthroline and the like have been employed commonly. From the viewpoints of sensitivity, specificity, solubility and so on, a metallochromic indicator for iron such as bathophenanthroline, 2-nitroso-5-(N-propyl-N-sulfopropylamino)-phenol (nitrosoPSAP), 3-(2-pyridyl)-5,6-bis[2-(5-furylsulphonic acid)]-1,2,4-triadine disodium salt, tripyridyl-triazine, ferrozine and the like has been used more commonly.
Since all of these metallochromic indicators can react only with bivalent iron, it is necessary to use a reducing agent in order to reduce trivalent iron when such metallochromic indicators are used. As a reducing agents for trivalent iron, L-ascorbic acid, thioglycolic acid, hydroxylamine hydrochloride, hydroquinone, hydrosulfite, sodium sulfite, hydrazine sulfate, metabisulfite (pyrosulfite) and the like have been known.
The entire iron atoms in serum exist in a form bound to transferrin, which is a type of serum globulins. Therefore, in order to measure the total concentration of iron in serum, it is necessary to release the iron from transferrin and to generate free iron before conducting measurement of iron concentration. As to a method of releasing the bound iron from transferrin, for example, the International Standard Method in which a protein removal method has been incorporated (Manual of Clinical Laboratory Method, ed. by Masamitsu Kanehara, rev. 32nd ed., p. 579, published in Feb. 20, 2005 by Kanehara & Co., Ltd.) and the Matsubara's modified method which has been emphasized as basic data during the process for establishing the International Standard Method (Manual of Clinical Laboratory Method, ed. by Masamitsu Kanehara, rev. 32nd ed., 1st issue, p. 580-581, published in Feb. 20, 2005 by Kanehara & Co., Ltd.) have been known. According to these methods, after removing protein in a sample to be measured for iron concentration, a metallochromic indicator for iron is added to the sample to measure absorbance, and thus the total concentration of iron in a sample can be measured.
By the way, in recent years, in association with the development of automated analyzers, clinical laboratory test is now being performed commonly using an automated analyzer. Use of such an automated analyzer is economical because volumes of specimen and reagent solution to be collected for the measurement can be made small, and has an advantage in capability of processing a large number of specimens at the same time. Accordingly, it is desired that the measurement of concentration of iron in serum or the like is also performed using an automated analyzer. However, since the International Standard Method and the Matsubara's modified method require a protein removal treatment prior to performing the measurement of iron, there is a problem that it is difficult to apply these methods to the measurement using an automated analyzer.
On the other hand, there is a method of measuring iron concentration called “direct calorimetric method” (hereinafter referred to as “direct method”), in which measurement of iron concentration is performed by adding a reducing agent and a metallochromic indicator directly to a sample (for example, non-patent reference 1: Manual of Clinical Laboratory Method, ed. by Masamitsu Kanehara, rev. 32nd ed., 1st issue, p. 579, published in Feb. 20, 2005 by Kanehara & Co., Ltd.). Since this method does not require the protein removal treatment in contrast to the above-described International Standard Method and the like, it can be applied to the system using an automated analyzer. Therefore, in the field of clinical laboratory test, the direct method of measuring iron concentration using an automated analyzer has been in widespread use.
However, there still remains a problem that the measured value obtained by the direct method using an automated analyzer is lower as compared to that obtained by the International Standard Method. In the International Standard Method, iron in a sample can be released completely (100%) from transferrin because sample is subjected to the protein removal treatment, while iron cannot be released completely from transferrin in the direct method using an automated analyzer, and it has been reported that percentage of the released iron (degree of release) is in a range from about 93 to 95% (the 51st plenary meeting of the Japanese Society of Laboratory Medicine, presentation No. 0-76, 2004), and this has been considered to be one of causes, which give rise to such problem as described above.
Therefore, under the current situation, establishment of a method of measuring iron concentration, which can provide the same level of measured value as that obtained by the International Standard Method and can be applied to the automated analyzer system, has been desired.
Non-patent Document 1: Manual of Clinical Laboratory Method, ed. by Masamitsu Kanehara, rev. 32nd ed., 1st issue, p. 579, published in Feb. 20, 2005 by Kanehara & Co., Ltd.